Fig 2: Close association of epigenetic status with Ets1 level.a ChIP-seq analysis of H3K27Ac and H3K27me3 at Ets1 promoter locus in MCF-7 (blue line) and MDA-MB-231 (red line) cells. b ChIP analysis of active marker (H3Ac) and inactive markers (H3K9me3 and H3K27me3) at Ets1 promoter locus under unstimulated or P/I stimulation conditions. The data from each replicate were normalized to the input control and the graphs represent fold enrichment of the indicated proteins to control antibody at the designated locus. Data are presented as mean ± SD. Two-way ANOVA with Bonferroni post-tests showed a significant difference of binding efficiency of H3Ac, H3K9me3, and H3K27me3 between MCF-7 and MDA-MB-231 cells. c DNA methylation status in the nine CpG sites (−375 or −534) was analyzed by bisulfite sequencing under unstimulated or stimulated (P/I) condition. Closed and open circles indicate methylated and unmethylated CpG sites, respectively. Bar graphs represent percentage methylation. Data are presented as mean ± SD. Two-way ANOVA with Bonferroni post-tests showed a significant difference of DNA methylation status. d Heatmap of RNA-sequencing data showing relative expression of DNA methylation-related and demethylation-related factors. e Analysis of relative expression levels in transcripts normalized against Hprt and proteins by qRT-PCR and Immuno-blots, respectively. Data are presented as mean ± SD. One-way ANOVA with Bonferroni correction indicated a significant difference of expression. f Scatterplots and nonparametric Spearman’s rank correlation (ρ) analysis with corresponding p-values correlation analysis from CCLE. Correlation between Ets1 and DNA methylation-related genes (Dnmt3a, Dnmt3b, and Apobec3c) in mRNA. Each symbol represents an individual human breast cancer cell line. Green circle: MCF-7 cells; Red circle: MDA-MB-231 cells. b, c, e Data shown are representative of more than two independent experiments with similar results. *p < 0.05, **p < 0.01, and ***p < 0.001 (unpaired t-test)

Fig 3: Ets1 exerts disease‐promoting effects in RMS and is under positive regulatory control of P3F in FP‐RMS. (A) Stable, shRNA‐mediated, depletion of Ets1 protein expression in RMS cells, as determined by protein immunoblotting with tubulin as loading control (n = 3, representative data shown). (B) Effects of Ets1 shRNA‐mediated depletion on clonogenic growth of RMS cells. Shown are representative images of colonies formed, and quantifications of colony count data. The latter are plotted as mean and standard error of the mean of three independent experiments (n = 3), each performed in triplicate, with the control set to 1; P‐values were determined using one‐way analysis of variance (1‐way ANOVA) with multiple comparisons; *P < 0.05, **P < 0.01. (C) Effects of Ets1 depletion on transendothelial invasion by RMS cells. Shown are representative images (scale bar = 50 µm), and quantifications, of invaded cells. Data show mean and standard error of the mean of three independent experiments (n = 3), each performed in duplicate, with the control set to 1; P‐values were determined using one‐way ANOVA with multiple comparisons, or two‐way Student's t‐test with unequal variance (Rh41 data); *P < 0.05, **P < 0.01. (D) Effects of Ets1 depletion on metastasis in the tail vein injection model. Scrambled control or shEts1 Rh30 FP‐RMS cells, each additionally expressing a luciferase reporter, were injected into the tail vein of NOD‐SCID/gamma mice [n = 10 (scrambled) and 9 (shEts1)]. Metastasis development was monitored using IVIS imaging following administration of luciferin. Top panel shows IVIS imaging data at end of experiment. Bottom panel shows IVIS quantification data (mean and standard error of the mean of photon flux; P‐value from 2‐way ANOVA with repeated measures). (E) Effects of PAX3/FOXO1 (P3F) depletion on KDM3A and Ets1 protein levels in FP‐RMS cells. Representative immunoblots and mean of densitometric quantifications from two independent experiments (n = 2), normalized to scrambled shRNA control (FOXO1 antibody was used for detection of P3F, as done previously [13]).

Fig 4: ETS1 directly stimulates miR-23a-3p transcription upon sorafenib treatment. A The expression of miR-23a-3p and B pri-miR-23a in MHCC97L and PLC/PRF/5 cells treated with sorafenib for 24 h. The doses of sorafenib were corresponding to 0, IC10, IC15, and IC20 values of sorafenib. C Venn diagram showing the intersections of data from proteomics analysis, ChIP-X, TransmiR and Circuit. Three TFs: ETS1, NFIC, and SP1 were the common TFs. D Gene ranking of potential TFs of proteomics data according to their log2FC. ETS1 was the highest upregulated TF. E The expression of miR-23a-3p in MHCC97L and F PLC/PRF/5 by qRT-PCR and the expression of ETS1 by immunoblotting. G Luciferase activity of pGL-23AP639 in HEK293. One-way ANOVA, p < 0.005***, p < 0.0001****. H The binding motif of ETS-1 on miR-23a promoter and the fold enrichment of fragments of miR-23a promoter was higher in sorafenib treated MHCC97L after the pulldown by ETS-1 Ab. The IgG Ab group was set as the negative control. Unpair t-test, p < 0.0001****

Fig 5: Transcription factor ETS1 regulated KRT80 expression and ETS1 is a target of miR-206 in ovarian cancer cell. (A) ChIP-PCR showed that ETS1 could bind the promoter region of KRT80 in ovarian cancer cells using ETS1 primary antibody, IgG was used as a negative control (n = 3). (B) ChIP-PCR products were analyzed using horizontal agarose gel electrophoresis and visualized using UV. (C) Correlation between KRT80 mRNA and ETS1 mRNA in 379 ovarian samples using starBase. (D) Binding site between miR-206 and 3ʹ-UTR of ETS1. (E) Dual Luciferase Gene Reporter System for detection of luciferase activity in ETS1-WT or ETS1-MUT and miR-206 mimic or miR-Mock co-transfection. (F) Representative images and quantitation of the western blotting showed that the protein expression of ETS1 in the miR-206 mimics/inhibitor groups (n = 3). GAPDH was used as an internal control. Data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.